Identification of mitogen-activated protein kinases substrates in Arabidopsis using kinase client assay

ABSTRACT Mitogen-activated protein kinase (MPK) cascades are essential signal transduction components that control a variety of cellular responses in all eukaryotes. MPKs convert extracellular stimuli into cellular responses by the phosphorylation of downstream substrates. Although MPK cascades are predicted to be very complex, only limited numbers of MPK substrates have been identified in plants. Here, we used the kinase client (KiC) assay to identify novel substrates of MPK3 and MPK6. Recombinant MPK3 or MPK6 were tested against a large synthetic peptide library representing in vivo phosphorylation sites, and phosphorylated peptides were identified by high-resolution tandem mass spectrometry. From this screen, we identified 23 and 21 putative client peptides of MPK3 and MPK6, respectively. To verify the phosphorylation of putative client peptides, we performed in vitro kinase assay with recombinant fusion proteins of isolated client peptides. We found that 13 and 9 recombinant proteins were phosphorylated by MPK3 and MPK6. Among them, 11 proteins were proven to be the novel substrates of two MPKs. This study suggests that the KiC assay is a useful method to identify new substrates of MPKs.


Introduction
Mitogen-activated protein kinase (MPK) cascades have been known to be highly conserved signal transduction modules in all eukaryotes including plants.MPK cascades are typically composed of three classes of protein kinases, MPK kinase kinase (MPKKK), MPK kinase (MPKK), and MPK.MPK cascades transduce extracellular signals to intracellular compartments to result in cellular response through the phosphorylation of downstream targets [1][2][3] .MPKs are activated by consecutive activation of MPKKKs and MPKKs in response to external signals.Activated MPKs directly phosphorylate the conserved motif of substrates, Ser/Thr residues followed by a Pro residue (S/T-P). 4,5hosphorylation of substrates by MPKs is known to generate appropriate cellular responses by changing subcellular localization, stability, transcriptional activity, and interaction with other proteins. 6,7][14] In uncovering the intricate roles of MPKs in plants, the identification of new substrates of MPKs is important.][17][18][19][20] However, it is believed that a large number of MPKs substrates have not been identified yet because the possible combinations of MPKKK-MPKK-MPK cascades are very complex, thereby the specific downstream substrates of possible combinations would be required to result in appropriate responses.
The kinase client (KiC) assay was developed as a new method for identifying the substrates of kinases through in vitro phosphorylation of synthetic peptide library coupled with tandem mass spectrometry. 21This method has potential application for high throughput characterization of kinasessubstrates interaction.Successfully, 23 proteins were identified as putative substrates of 17 different protein kinases using the KiC assay. 22In addition, novel substrates of P2K1 (DORN1) and ILK1 were identified by the KiC assay. 23,24Eventually, the biological and biochemical functions of RBOHD and ILK5 isolated by the KiC assay were extensively investigated as new substrates of P2K1. 24,25In this study, we identified potential substrates of MPK3 and MPK6 by the KiC assay.Furthermore, these peptides were verified through in vitro kinase assay with recombinant proteins.As a result, we confirmed that a half of potential substrates were phosphorylated by MPKs.Finally, we identified 11 novel substrates of MPKs that would provide clues for understanding MPK-mediated signaling in Arabidopsis.Conclusively, this research suggests that the KiC assay is a useful tool for the identification of novel MPK substrates in plants.

Construction of synthetic peptide library
Based on the results obtained from in vivo phosphoproteomic analysis in Arabidopsis thaliana, a library (PEPscreen, sigma, St. Louis, MO, USA) consisting of approximately 2,100 10 to 20-mer peptides was synthesized.Stock peptide solutions were prepared by dissolving the peptides in 80% (v/v) dimethylformamide in water to a final concentration of 8 mM.Samples from the stock solutions were then diluted into the KiC assay. 21,26These synthetic peptides were then used as the basis for an integrated experimental strategy for the identification of kinase-client proteins.

Expression of recombinant his-MPK3 and MPK6 in E. coli
Full-length MPK3 and MPK6 cDNA were amplified by PCR from a cDNA library of Arabidopsis seedlings using gene-specific primers (Table S1) and subcloned into pQE30 for the expression of 6×His-fused MPK3 and MPK6.The 6×His-tag fusion proteins were expressed in Escherichia coli (M15) and purified using Ni-NTA agarose beads (Qiagen, Hilden, Germany).

Phosphorylation of synthetic peptides by MPK3 and MPK6
The KiC assay was performed as previously described with slight modifications. 26In vitro kinase reaction of a mixture of synthetic peptides containing 2,100 different peptides was conducted using recombinant MPK3 or MPK6 at 37°C for 1 h with shaking.Kinase reaction was stopped by adding one volume of 1% formic acid/99% acetonitrile, evaporated to near dryness in a centrifugal evaporator, and then stored at −20°C until mass spectrometry.

Liquid chromatography and mass spectrometry
Freeze-dried peptides were dissolved by adding 40 µL of 0.1% formic acid.Samples were loaded into 96-well plates which were then placed onto a pre-chilled 10°C autosampler.10 µL of each sample was analyzed using Finnigan Surveyor liquid chromatography (LC) system attached to either a stand-alone LTQ-XL or a LTQ Orbitrap XL ETD mass spectrometer (Thermo Fisher, San Jose, CA, USA).Peptides are separated on a C18 microcapillary column using a mobile phase.
Analysis of the synthetic peptides screen was performed using a stand-alone LTQ-XL.Analysis of the recombinant protein kinase client assay was performed using a LTQ Orbitrap XL ETD.The mass spectra were collected using nanospray ionization in the positive ion mode.Detailed mass spectrometry was performed as previously described. 22Data-dependent ions fragment with ETD has a mass exclusion width of 10 ppm.Decision tree settings were previously described. 27The reagent ion source settings, including temperature, emission current, energy level, and CI pressure were 160°C, 50 µA, −70 V, and 17.5 psi, respectively.The activation time was 100 ms and supplemental activation mode was enabled.

Bioinformatics analysis
The raw MS files were searched against a decoy database consisting of the random complement of the sequences comprising the peptide library, using SEQUEST algorithms (Proteome Discoverer 1.0, Thermo Fisher).Instrument and search parameter settings have been previously described. 22,26Identification data were evaluated using the XCorr function of SEQUEST, and phosphorylation-site localization was accomplished using phosphoRS (Proteome Discoverer, v. 1.0.3,Thermo Fisher).The XCorr values for each charge state were set to default and no decoy hits were allowed.Peptide mass deviation was 10 ppm and a setting of two PSMs/protein was used to further filter the data.For final validation, each spectrum was inspected manually and accepted only when the phosphopeptide has the highest pRS site probability, pRS score, XCorr value, and site-determining fragment ions allowed unambiguous localization of the phosphorylation site.Phospho-peptides with a pRS score ≥ 50 and/or a pRS site probability of ≥ 55% were accepted.

Expression of recombinant MPK3 and MPK6 substrate proteins in E. coli
Full-length cDNAs of substrate were amplified by PCR from a cDNA library of Arabidopsis seedlings using genespecific primers (Table S1) and cloned into the pGEM-T Easy Vector (Promega, USA).Verified inserts by sequencing were excised with proper restriction enzymes and subcloned into pGEX 5X-1 for the expression of Glutathione S-transferase (GST)-fused proteins.GSTfusion proteins were expressed in E. coli BL21(DE3) and purified using glutathione Sepharose-4B beads (GE Healthcare, Piscataway, NJ, USA).

Autophosphorylation site mapping of recombinant MPKs produced in E. coli
MPKs are known to be fully activated by the phosphorylation of Thr-X-Tyr (T-X-Y) motif in activation loop by upstream MPKKs. 29Since many studies showed that recombinant MPKs produced in E. coli had basal activity, 9,11,15,30 in this study, we performed the KiC assay by using recombinant MPKs purified from E. coli without the activation by MPKKs.Expectedly, purified recombinant MPKs had basal kinase activities.These results indicate that recombinant MPKs produced in E. coli may have basal activities through autophosphorylation.However, the autophosphorylation sites involved in the basal activations of MPKs are poorly identified.
Therefore, we analyzed the autophosphorylation site of recombinant MPK3 and MPK6 that were produced in E. coli in the absence or presence of ATP.Quantitative mass spectrometry revealed that autophosphorylation occurred on multiple residues of recombinant MPKs (Figure 1).Unexpectedly, T-X-Y motif in MPK3 (Thr 196 and Tyr 198 ) and MPK6 (Thr 221 and Tyr 223 ) were already autophosphorylated before the addition of ATP.Interestingly, the autophosphorylations of Thr 196 and Tyr 198 residues in MPK3 and Tyr 223 residue in MPK6 significantly enhanced by the addition of ATP (Figure 1).Furthermore, Tyr, 37 Thr 58 , Ser 157 , Tyr 274 , Ser 291 and Ser 406 residues of MPK3 also were autophosphorylated only in the presence of ATP (Figure 1).This result indicates that recombinant MPKs produced in E. coli have their basal activities through autophosphorylation.  *Amino acid residues that could potentially be phosphorylated by MPK3.b pRS score is based on the cumulative binomial probability that the observed match is a random event.Potential phosphorylation sites were predicted by pRS score.c pRS site probabilities are estimations of the probability (0 ~ 100%) for the respective site being truly phosphorylated.

Identification of potential substrates of MPK3 and MPK6 using the KiC assay
To identify novel putative substrates of MPK3 and MPK6, we performed the KiC assay according to the standard procedure. 26Purified recombinant MPK3 or MPK6 was incubated with a peptide library comprised of approximately 2,100 synthetic peptides (representing in vivo phosphorylation events) and phosphorylated peptides were analyzed by tandem mass spectrometry.As results, we identified 23 and 21 peptides as potential client peptides of MPK3 (Table 1) and MPK6 (Table 2), respectively.Among these client peptides, eight client peptides (AT1g07630, AT1g15400, AT1g53050, AT2g38280, AT3g04470, AT4g02300, AT4g12770, and AT4g42590) were isolated by both MPK3 and MPK6, whereas 15 and 13 client peptides were specifically isolated by either MPK3 and MPK6, respectively (Figure 2A).

Nonconserved motifs are phosphorylated by MPKs in the KiC assay
It is well reported that MPKs mostly phosphorylate their substrates within a simple conserved motif, Ser or Thr residues followed by Pro residue (S/T-P). 4,5In this study, we confidently identified phosphorylation sites of isolated client peptides by tandem mass spectrometry (Tables 1,2).Almost   all mapped client peptides were phosphorylated on either Ser or Thr residues.However, the Tyr residues of some client peptides were surprisingly phosphorylated by MPK3 and MPK6 (Figure 3a).Meanwhile, four and eight isolated client peptides were phosphorylated on the conserved motif by MPK3 and MPK6, respectively.However, 19 and 13 isolated client peptides were unexpectedly phosphorylated within nonconserved motifs by MPK3 and MPK6, respectively (Figure 3b).These results suggest that MPKs can phosphorylate nonconserved Ser or Thr residues and even Tyr residues in in vitro reconstituted experiments.

Verification of the substrates using recombinant fusion protein
Because the client peptides in the KiC assay were 10-to 20-mer short synthetic peptides, it is required to verify whether the fulllength fusion proteins of isolated client peptides can also be phosphorylated by MPKs.To examine the phosphorylation of full-length proteins by in vitro kinase assay, we expressed and purified GST-fused full-length proteins of isolated client peptides.
In the case of AT3g62700, we constructed a partial cDNA clone encoding the partial fusion protein including the potential phosphorylation site because we failed to produce its full-length fusion protein.As results, we found that 13 of 23 fusion proteins were phosphorylated by MPK3 (Figure 4A).Similarly, 9 of 21 fusion proteins were phosphorylated by MPK6 (Figure 4B).Overall, these results demonstrate that approximately half of client peptides isolated by the KiC assay were verified as the potential substrates of MPKs in vitro.Interestingly, 4 of 13 isolated substrate proteins have already been reported as MPK3 substrates (Table 3) and 7 of 9 isolated substrate proteins were already reported as MPK6 substrates (Table 4).Among these substrates, three substrates (AT1g15400, AT2g38280, and AT4g12770) were phosphorylated by both MPK3 and MPK6, while five substrates (AT1g08420, AT1g11360, AT3g55270, AT4g39680, and AT5g20280) were specifically phosphorylated by either MPK3 or MPK6.][17][18][19][20] The phosphorylation sites of four substrates were same with those previously reported, but the phosphorylation sites of four other substrates were different from those previously reported (Table S2).These results indicate that not only the same phosphorylation sites but also the new phosphorylation sites can be identified from the same substrates by the KiC assay.Conclusively, based on the KiC assay we could identify nine and two novel putative substrates of MPK3 and MPK6, respectively.

Identification of new substrates of MPKs using the KiC assay
2][3] To understand the novel functions of MPK cascades in many signaling pathways, isolation of new substrates of MPKs is essential.In this study, we applied the KiC assay to identify novel substrates of MPKs.As a result, 23 and 21 client peptides were identified as putative substrates of MPK3 and MPK6, respectively (Tables 1, 2).Interestingly, eight of identified client peptides were overlapped (Figure 2A), indicating that the targets of MPK3 and MPK6 are redundant in some signaling pathways.13 client peptides of MPK3 and 9 client peptides of MPK6 were verified as substrates of MPKs by in vitro kinase assay of their fusion proteins.In conclusion, we identified nine novel   1 and 2).MBP and GST were used as positive and negative control, respectively.
substrates of MPK3 and two novel substrates of MPK6 in this study (Figure 4).Using the same KiC assay, 23 putative substrates of P2K1 were identified and some of them were extensively studied. 24Conclusively, the new signaling pathway of plant immune response controlled by P2K1 was elucidated by the phosphorylation studies of those isolated substrates. 24,25,32hus, we suggest that the KiC assay is a useful method to identify novel substrates of MPKs and the novel signaling pathway of MPKs through these substrates may be revealed by subsequent research.

Validation of the KiC assay using in vitro kinase assay
Generally, it is known that MPKs could phosphorylate the Ser or Thr residues on the S/T-P motif of their substrates. 4,5nterestingly, most isolated client peptides were phosphorylated on Ser or Thr residues, but two isolated client peptides (AT5g13240 and AT5g42590) were phosphorylated on Tyr residues by the KiC assay (Figure 3A).However, subsequent in vitro kinase assay of fusion proteins showed that these recombinant proteins were not phosphorylated by MPKs (Figure 4).These results indicate that the KiC assay results included nonspecific phosphorylation.Since we confirmed isolated client peptides by the KiC assay through in vitro kinase assay of their recombinant proteins (Figure 4), the novel substrates identified in this study would be bona fide substrates of MPKs.
We speculate that nonspecific phosphorylation in the KiC assay is possibly due to the excess amount of peptides and kinases employed.To improve the accuracy of the KiC assay, it is necessary to obtain results by comparing a series of different dilution reactions of peptides and kinases.Through this series of reactions can minimize nonspecific phosphorylation while maintaining efficient phosphorylation of substrates.

Further research of novel identified MPKs-substrate modules
In this study, we identified 11 novel substrates of MPKs by in vitro kinase assay (Figure 4, Table 3, 4).5][36][37] To identify the biological roles of their phosphorylation by MPKs, it is necessary to test whether the phenotypes of these knock-out mutants can be rescued by the expression of the non-phosphorylation form.In the case of unknown substrates (AT2g04235, AT2g21300, AT3g04470, AT3g19330, and AT4g18950), the biological function of them would be identified first, and then the biological function of their phosphorylation should be investigated.In this report, we mainly focused on the usefulness of the KiC assay for the identification of novel substrates of MPKs by the assay in vitro.Therefore, independent more detail functional and biochemical researches including in vivo phosphorylation of the identified substrates by MPKs should be performed.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.Autophosphorylation sites of recombinant MPK3 and MPK6.Quantitative mass spectrometry for identified phosphorylated sites of recombinant (a) MPK3 and (b) MPK6.Spectral count data presented are means of three replicates.Dark and white bars represent the presence or absence of ATP, respectively.

Figure 2 .
Figure 2. Venn diagram analysis of MPKs client peptides identified by the KiC assay.(a) Venn diagram showed the overlap between the identified MPK3 and MPK6 client peptides.(b) Venn diagram showed the overlap between the identified MPK3 client peptides and previously published datasets.(c) Venn diagram showed the overlap between the identified MPK6 client peptides and previously published datasets.The numbers 325 and 2,209 represent the number of reported substrates of MPK3 or MPK6 by various methods, respectively (Figure S1) 15-20 .The overlap segments indicate the numbers of the same substrates.

Figure 4 .
Figure 4. Verification of putative substrates using in vitro kinase assay.In vitro kinase performed with (a) 6XHis-MPK3 (2 µg) or (B) 6XHis-MPK6 (2 µg) and the recombinant substrate protein (3 µg) in the presence of [γ-32 P] ATP.After SDS-PAGE, the proteins were visualized by Coomassie blue staining (Staining).The phosphorylated proteins were visualized by autoradiography (Autorad).The asterisks represent phosphorylated substrate proteins, and the arrow represents the position of substrate proteins in SDS-PAGE.The numbers represent the fusion protein of MPK3 or MPK6 client peptides (Tables1 and 2).MBP and GST were used as positive and negative control, respectively.

Table 2 .
List of identified client peptides by KiC assay using a synthetic peptide library with recombinant MPK6.
b pRS score is based on the cumulative binomial probability that the observed match is a random event.Potential phosphorylation sites were predicted by pRS score.

Table 3 .
List of phosphorylated recombinant substrates by MPK3 using in vitro kinase assay.The number of + indicates the intensity of phosphorylation by MPK3, and -indicates no phosphorylation by MPK3.c Protein subcellular localization extracted from TAIR database. b

Table 4 .
List of phosphorylated recombinant substrates by MPK6 using in vitro kinase assay.
cProtein subcellular localization extracted from TAIR database.